Biology Reference
In-Depth Information
scattering) of the protein to calculate protein concentration, compared with the lipid
concentration determined from a phosphate assay (
Chen, Toribara, &Warner, 1956
).
It is also possible to use protein assays such as the bicinchoninic acid (BCA) assay or
other comparisons with bovine serum albumin, but these methods are less accurate.
18.2
FRET MEASUREMENTS AND ANALYSIS
FRET is an excellent technique for determining protein-protein interaction but care
must be taken over the interpretation of results. FRET is the nonradiative transfer of
energy between a donor fluorophore and an acceptor fluorophore. For a review of
different fluorescence techniques for studying GPCR interactions, see
Goddard
and Watts (2012)
. FRET is distance-dependent (
R
6
) and as such has a defined win-
dow of operation that is suitable for biological measurements (
Stryer, 1978
). The ef-
ficiency of FRET between fluorophores is characterized by the F
¨
rster distance (
R
0
),
which is the distance at which energy transfer is 50% efficient. Typically, it is only
possible to measure FRET if the separation of the fluorophores is within 25% of the
F
¨
rster distance. For example, the
R
0
of the CFP-YFP pair is 49
˚
. When CFP or
YFP are linked to a receptor via a flexible linker, this will allow FRET in almost
any receptor conformation (assuming a receptor diameter of
40
˚
). However, care
must be taken when using small molecule fluorophores—if these have short
R
0
values and are placed on distant parts of the dimer, dimerization may occur without
the production of FRET.
Taking NTS1-CFP and NTS1-YFP as an example, several controls need to
be generated and assayed. Empty liposomes must be produced to account for
scattering and any endogenous fluorescence, which normally arises due to
contamination of the solvents used in preparation of the lipids. Liposomes
containing NTS1-CFP or NTS1-YFP alone should also be generated and, finally,
a sample containing equimolar amounts of NTS1-CFP and NTS1-YFP. The
measurements illustrated in
Table 18.1
should then be made using an appropriate
Table 18.1
FRET Measurements
Sample
Excitation (nm)
Emission Spectrum (nm)
1.
Liposomes
440
450-600
2.
Liposomes
510
520-600
3.
NTS1-CFP
440
450-600
4.
NTS1-YFP
440
450-600
5.
NTS1-YFP
510
520-600
6.
NTS1-CFPþNTS1-YFP
440
450-600
7.
NTS1-CFPþNTS1-YFP
510
520-600
Fluorescence measurements should be taken for the indicated samples and excitation and emission
wavelengths. Adjust wavelength appropriately if using alternative fluorophores.
